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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [2]
- Immunohistochemistry [2]
- Flow cytometry [3]
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- Product number
- PA5-95343 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- SPTLC1 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Reconstitute with 0.2 mL of distilled water to yield a concentration of 500 µg/mL. Positive Control - WB: human Hacat whole cell, human A431 whole cell, human SIHA whole cell, human U251 whole cell. IHC: human intestinal cancer tissue. ICC/IF: U20S cell. Flow: U20S cell.|Store at -20°C for one year from date of receipt. After reconstitution, at 4°C for one month. It can also be aliquotted and stored frozen at -20°C for six months. Avoid repeated freeze-thaw cycles.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 500 µg/mL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of SPTLC1 in, Lane 1: human placenta tissue lysates, Lane 2: human U-87MG whole cell lysates, Lane 3: human A431 whole cell lysates, Lane 4: human PC-3 whole cell lysates, Lane 5: human U20S whole cell lysates, Lane 6: human HepG2 whole cell lysates, Lane 7: human U-937 whole cell lysates. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 µg of sample under reducing conditions. After Electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. The membrane was blocked with 5% non-fat milk/TBS for 1. 5 hour at RT. The membrane was incubated with SPTLC1 Polyclonal Antibody (Product # PA5-95343) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0. 1% Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5,000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit. A specific band was detected for SPTLC1 at approximately 60 kDa. The expected band size for SPTLC1 is at 53 kDa.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of SPTLC1 using SPTLC1 Polyclonal Antibody (Product # PA5-95343). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel)/90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 µg of sample under reducing conditions. Lane 1: human Hacat whole cell lysates. Lane 2: human A431 whole cell lysates. Lane 3: human SIHA whole cell lysates. Lane 4: human U251 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with SPTLC1 Polyclonal Antibody at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5,000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for SPTLC1 at approximately 53 kDa. The expected band size for SPTLC1 is at 53 kDa.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of SPTLC1 in U20S cell. Antigen retrieval was performed on the tissue using IHC enzyme antigen retrieval reagent and blocked with 10% goat serum. Samples were incubated with SPTLC1 polyclonal antibody (Product # PA5-95343) at a 2 µg/mL dilution, followed by 488 conjugated goat anti-rabbit IgG using a 1:100 dilution and DAPI.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of SPTLC1 in U20S cell. Antigen retrieval was performed on the tissue using IHC enzyme antigen retrieval reagent and blocked with 10% goat serum. Samples were incubated with SPTLC1 polyclonal antibody (Product # PA5-95343) at a 2 µg/mL dilution, followed by 488 conjugated goat anti-rabbit IgG using a 1:100 dilution and DAPI.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of SPTLC1 in paraffin-embedded human intestinal cancer tissues. Antigen retrieval was performed on the tissue using citrate buffer (pH 6, 20 min) and blocked with 10% goat serum. Samples were incubated with SPTLC1 polyclonal antibody (Product # PA5-95343) at a 1 µg/mL dilution, followed by biotinylated goat anti-rabbit IgG (30 min, 37°C), and developed with Strepavidin-Biotin-Complex and DAB.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of SPTLC1 in paraffin-embedded human intestinal cancer tissues. Antigen retrieval was performed on the tissue using citrate buffer (pH 6, 20 min) and blocked with 10% goat serum. Samples were incubated with SPTLC1 polyclonal antibody (Product # PA5-95343) at a 1 µg/mL dilution, followed by biotinylated goat anti-rabbit IgG (30 min, 37°C), and developed with Strepavidin-Biotin-Complex and DAB.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry of SPTLC1 in U2OS cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with SPTLC1 Polyclonal Antibody (Product # PA5-95343) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry of SPTLC1 in U2OS cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with SPTLC1 Polyclonal Antibody (Product # PA5-95343) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow cytometry analysis of SPTLC1 in U20S cells using SPTLC1 Polyclonal Antibody (Product # PA5-95343), shown in overlay histogram (blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum, and incubated with the primary antibody (1 μg/1x10^6 cells) for 30 min at 20°C. DyLight 488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
